The present invention relates to a novel activity of template-dependent polymerases and, more particularly, to the incorporation of oligonucleotide triphosphates in a template-dependent manner onto a growing nascent oligonucleotide-3′-OH group by such polymerases, to methods exploiting the advantages of the novel activity, to compositions for implementing the methods and to compounds generated while implementing the methods. The present invention provides a novel platform technology, which can be used to develop novel nucleic acid-based applications for biotechnology and nanotechnology including, for example, pharmaceutics, biocatalysis and diagnostics.
It is well recognized that nucleic acid polymers possess functional capacities. These qualities may be exemplified in vivo as specific recognition of tRNA anticodons during translation, and by splicing activity of ribozymes. In vitro, several systems have been established from which functional nucleic acid polymers can be isolated. These methods of in-vitro evolution, termed hereinafter the directed evolution approach, include SELEX (systematic evolution of ligands by exponential enrichment) of RNA (Beaudry & Joyce, 1992) and DNA (Breaker & Joyce, 1994), and iterative use of combinatorial libraries of oligonucleotides (Frank, 1995).
In spite of their poor number of functional groups (i.e., four bases in natural nucleotides), nucleic acid polymers may yield diverse activities such as specific binding affinity to a target molecule or catalysis of chemical-bonds formation. Recently, the inclusion of nucleotide analogs bearing alternative combination of functional groups further extend the vocabulary of nucleic acids, and establish enzymatic approaches for directed evolution as efficient technologies for isolation of functional polymers (Eaton, 1997; Benner et al., 1998; Earnshaw & Gait, 1998).
Naturally-occurring nucleic acid polymers (DNA and RNA) maintain their basic information in the sequence order and combination of four distinct nucleotides, identified by their nitrogenous base moieties adenine and guanine, which are purine derivatives, and cytosine and thymidine (for DNA) or uracil (for RNA), which are pyrimidine derivatives (see FIG. 1).
Information transfer (e.g., DNA-dependent DNA replication, DNA-dependent RNA transcription, RNA-dependent DNA reverse transcription and RNA-dependent RNA replication) is performed enzymatically by mirror copying of the sequence combination in one polymer to a new polymer according to a binary code known as complementation, wherein an adenine nucleotide is complementary to a thymidine nucleotide (or uracil nucleotide) and vice versa, whereas a guanine nucleotide is complementary to a cytosine nucleotide and vice versa.
The genetic binary code, which stores genome information in all organisms over time, entails a simple information transfer key based on electrostatic and steric complementation between two pairs of matching nucleotides. This code has been optimized by natural evolution as advantageous for reliable transfer of genetic information between generations of organisms, between cells within an organism, and between certain complexes and compartments within cells. For example, genetic information is transferred in eukaryotes when DNA stored in the nucleus is transcribed to RNA, which is then translocated to the cytoplasm and translated by the ribosomal machinery to polypeptides.
At the down of evolution, the relatively low complexity of nucleic acid may have been sufficient for the emergence of some activities that were probably limited to assembly and cleavage of nucleic acids. Some of these functions are still exercised today in processes such as splicing and transposition. Later on in evolution, the low complexity of the binary code was mainly utilized for transfer and maintenance of genetic information, while on top of it, a more complex code was developed that dictates synthesis of additional polymers with enhanced complexity—the proteins. These polymers are coded by groups of three successive building blocks of nucleic acids, known as triplet codons, which are recognized and decoded by the ribosomal protein-translation machinery. By evolving the triplet codons, a relatively simple information code in one polymer can be translated and amplified into a new polymer with versatile and wide functional space. The increase in functional capacity may have been a major breakthrough in evolution developments leading to more advanced molecules and organisms.
While conceiving the present invention it was realized that should template-dependent polymerases be able to employ oligonucleotide triphosphates, instead of, or in addition to, nucleotide triphosphates as basic building blocks or units for template-dependent synthesis, the ability to create highly complex polymers having precisely locatable functional groups, and thereby better exploiting the information transfer capacity of nucleic acids in an unprecedented manner exceeding that of nature, will become available.
Assume, for example, the sole use of dinucleotide triphosphates as building blocks for a template-dependent synthesis of a nucleic acid molecule. Sixteen (24) different dinucleotide triphosphates are available for such synthesis, which represent all of the possible combinations of the four natural nucleotide monomers arranged as dimers. The 16 available dinucleotide triphosphates are: AA-triphosphate (SEQ ID NO:1); AC-triphosphate (SEQ ID NO:2); AG-triphosphate (SEQ ID NO:3); AT-triphosphate (SEQ ID NO:4); CA-triphosphate (SEQ ID NO:5); CC-triphosphate (SEQ ID NO:6); CG-triphosphate (SEQ ID NO:7); CT-triphosphate (SEQ ID NO:8); GA-triphosphate (SEQ ID NO:9); GC-triphosphate (SEQ ID NO:10); GG-triphosphate (SEQ ID NO:1); GT-triphosphate (SEQ ID NO:12); TA-triphosphate (SEQ ID NO:13); TC-triphosphate (SEQ ID NO:14); TG-triphosphate (SEQ ID NO:15); and TT-triphosphate (SEQ ID NO:16).
Further assume that unique functional groups are attached to some or all of the dinucleotide triphosphates building blocks. In this case, a polymer can be synthesized having a maximum of 16 available and precisely locatable types of functional groups, instead of a maximum of only four such groups. It will be appreciated that the maximal number of unique and precisely locatable functional groups depends on the number of monomers employed per oligonucleotide triphosphate. This maximal number equals 4N, where N is the number of monomers per oligonucleotide triphosphate.
Therefore, the use of oligonucleotide triphosphates by template-dependent polymerases, instead of, or in addition to, nucleotide triphosphates as basic building blocks or units for template-dependent synthesis, makes possible the creation of highly complex polymers having precisely locatable functional groups.
Furthermore, if the use of oligonucleotides as building blocks for nucleic acid synthesis will become feasible, it will be appreciated that each building block becomes scarcer as compared to the use of nucleotide triphosphates. This phenomenon increases with length (N) of the oligonucleotides employed. Thus, assuming equal representation for each of the four nucleotides in a given nucleic acid polymer, a particular mononucleotide is expected, statistically, every 4 nucleotides in this polymer, a dinucleotide is expected every 16 nucleotides, a trinucleotide every 64 nucleotides (see Table 1, below), a tetranucleotide every 256 nucleotides, a pentanucleotide every 625 nucleotides, and an oligonucleotide of N-mer is expected every 4N nucleotides, in the nucleic acid polymer. Consequently, while using relatively short oligonucleotide sequences as building blocks for template-dependent nucleic acid synthesis, not only the total number of building blocks required for synthesizing a given nucleic acid sequence is reduced, but also each building block is less represented. As is further exemplified below, this feature can be advantageously exploited in detection of nucleic acid sequences and related applications through template-dependent polymerization.
TABLE 1Nucleotide trimers can be arranged in 64 distinct combinations(SEQ ID NOs: 17-80, from left to right, top to bottom)AAA AAC AAG AATACA ACC ACG ACTAGA AGC AGG AGTATA ATC ATG ATT CAA CAC CAG CATCCA CCC CCG CCTCGA CGC CGG CGTCTA CTC CTG CTT GAA GAC GAG GATGCA GCC GCG GCTGGA GGC GGG GGTGTA GTC GTG GTT TAA TAC TAG TATTCA TCC TCG TCTTGA TGC TGG TGTTTA TTC TTG TTT
Previously, dinucleotides were indicated to be involved in initiation of transcription by RNA polymerase (Shaw et al., 1980), or as building-block units in assembly of oligonucleotide through non-enzymatic means (Leberton et al., 1993; Ordoukhanian & Taylor, 1997; Schmidt et al., 1997). In addition, modified dinucleotides have been used as inhibitors of various viral enzymes such as reverse transcriptase (Jahnke et al., 1995; Jahnke et al., 1997) and integrase (Taktakishvili et al., 2000). However, dinucleotide triphosphates and oligonucleotide triphosphates have not been shown to be involved, to our knowledge, in relation with template-dependent enzymatic polymerization of nucleic acids.
Therefore, there is a widely recognized need for, and it would be highly advantageous to have, methods for better exploiting the information transfer capabilities of nucleic acids (Schmidt et al., 1997; Koppitz et al., 1998; Ogawa et al., 2000), which can serve as a platform technology for development of molecules with novel biological activities, and for the development of novel nucleic acid amplification and identification schemes. Other applications and advantages of these methods will become apparent to those of skills in the art while reading the following sections of the specification.